Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro

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Abstract

Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm
communities; however, there are few studies demonstrating the role of biofilms
in chronic wound pathogenesis. This study establishes a novel method for
studying the effect of biofilms on the cell types involved in wound healing. Cocultures
of Staphylococcus aureus biofilms and human keratinocytes (HK) were
created by initially growing S. aureus biofilms on tissue culture inserts then transferring
the inserts to existing HK cultures. Biofilm-conditioned medium (BCM)
was prepared by culturing the insert-supported biofilm in cell culture medium. As
a control planktonic-conditioned medium (PCM) was also prepared. Biofilm,
BCM, and PCM were used in migration, cell viability, and apoptosis assays.
Changes in HK morphology were followed by brightfield and confocal microscopy.
After only 3 hours exposure to BCM, but not PCM, HK formed dendritelike
extensions and displayed reduced viability. After 9 hours, there was an increase
in apoptosis (p 0.0004). At 24 hours, biofilm-, BCM-, and PCM-exposed
HK all exhibited reduced scratch closure (p 0.0001). The results demonstrated
that soluble products of both S. aureus planktonic cells and biofilms inhibit
scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability
and significantly increased HK apoptosis compared with planktonic S. aureus.
Keratinocytes are the major cell type of the epidermis,
which serves as the primary barrier between the external
environment and the internal tissues. In this capacity,
the epidermis also functions as a barricade to microorganisms,
toxins, and various antigens. When the barrier is
breached due to wounding, basal keratinocytes from the
wound edges or dermal appendages migrate over the open
wound to reestablish the barricade in a process called reepithelialization.
Chronic wounds, such as diabetic foot
ulcers, venous leg ulcers, and pressure ulcers, are characterized
by prolonged inflammation, an altered wound matrix,
and the failure to reepithelialize.
Chronic wounds are also characterized as supporting a diverse
microbial flora. A literature review by Bowler examined
culture data from 62 published studies dating between
1969 and 1997.1 The most predominant wound isolate in
both chronic and acute wounds was Staphylococcus aureus
(reported in 63% of the studies), followed by coliforms
(45%), Bacteroides spp. (39%), Peptostreptococcus spp.
(36%), Pseudomonas aeruginosa (29%), Enterococcus spp.
(26%), and Streptococcus pyogenes (13%).1 Using molecular
techniques, Dowd et al.2 also demonstrated vast bacterial
diversity within chronic wounds. The most prevalent species
included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter,
Stenotrophomonas, Finegoldia, and Serratia spp.
It has been speculated that bacteria colonizing chronic
wounds exist as a biofilm.3–7 Biofilms represent bacterial
communities surrounded by extracellular polysaccharide
matrix. Such communities are often polymicrobial and resistant
to antimicrobials. Chronic wounds are an ideal environment
for bacterial infection and biofilm formation. The
wound remains open for a prolonged period of time, increasing
the odds of bacterial infection. The wound bed provides a
surface for growth, and poor blood flow and hypoxia discourage
native defenses.8 Studies have shown that wounds
inoculated with bacteria form biofilms.6,9 Furthermore, in a
recent study by James et al.,10 60% of chronic wound specimens
were characterized as containing biofilm compared
with 6% of acute wound specimens. Despite the prevalence
of biofilms in wounds, there are few data illustrating the
role of biofilms in chronic wound pathogenesis. This study
establishes a novel method for directly studying the effect
of biofilms on the cell types involved in wound healing.
Specifically, it examines the effect of S. aureus biofilms on
keratinocyte morphology, viability, and scratch closure.
METHOD ANDMATERIALS
Cell culture
Normal human keratinocytes (HK) were isolated from
newborn foreskin using methods previously described11
and in accordance with the University of Washington
690 Wound